1. Field of the Invention
A method is provided for simultaneously detecting affinity and specificity of a ligand or compound to serum protein albumin by using a thermal shift assay and a site-specific competition assay.
2. Description of the Related Art
Albumin is an abundant plasma protein, accounting for about 60% of total proteins in plasma. One of the main physiological functions of albumin is the transport of non-esterified fatty acids in the circulatory system. Albumin is also a general transporter protein for various endogenous ligands, including metal ions, bilirubin, bile salts, tryptophan, hormones, and vitamins. In addition, albumin binds to a wide range of therapeutic drugs or compounds (review in Kragh-Hansen et al., Biol Pharm Bull 25: 695-704, 2002).
As therapeutic drugs bind to albumin in plasma and reach the target tissues in a bound form, the binding or affinity to albumin greatly affects the pharmacological properties of therapeutic drugs in plasma. For example, the binding to albumin reduces the concentration of free active therapeutic drugs in plasma. Alternatively, the binding to albumin promotes the solubilization of hydrophobic compounds, buffers the distribution of drugs through the body, and reduces metabolic clearance of drugs. Therefore, albumin is routinely used to screen and characterize potential candidate compounds from the early phase of drug discovery to the optimization process in medicinal chemistry (reviewed in Colmenarejo, Med Res Rev 23: 275-301, 2003; Kragh-Hansen et al., Biol Pharm Bull 25: 695-704, 2002).
Albumins of different species share highly conserved sequences, structures, and properties. Human serum albumin (HSA) is a protein with a monomeric molecular weight of about 67 kDa. Crystallographic studies have indicated that HSA has three domains I, II, and III, and two sub-domains A and B within each of the domains (Carter and Ho, Adv Protein Chem 45: 152-203, 1994). Competitive binding studies have shown that HSA has two primary binding sites I and II, and several secondary binding sites (Sudlow et al., Mol Pharmaco 11: 824-832, 1975). The binding site I, located at subdomain HA, is larger and more flexible than the binding site II located at subdomain IIIA. The drugs or ligands have affinity to site I are mostly bulky heterocyclic anions, and the drugs or ligands have affinity to site II are mainly aromatic carboxylates. Subsequent studies show that endogenous ligands also have affinity to sites I and/or II (Kragh-Hansen et al., Biol Pharm Bull 25: 695-704, 2002; Curry et al., Biochimi Biophys Acta 1441: 131-140, 1990). Additionally, the binding of one ligand or compound at one site may affect the binding at other sites.
Several methods such as equilibrium dialysis, probe displacement, ultracentrifugation, and thermal shift assay have been used to detect formation of a ligand-albumin complex. These conventional methods analyze either the affinity or the specificity. To characterize both affinity and specificity properties of ligand-albumin binding, additional procedures, longer process time, more reagents and equipment, higher cost are required.
ThermoFluor® is a newly developed system for rapid thermal shift assay as described in Pantoliano et al. (J Biomol Screen 6: 429-440, 2001); Matulis et al. (Biochemistry 44: 5258-5266, 2005); U.S. Pat. Nos. 6,020,141; 6,036,920; 6,214,293; 6,232,085; 6,268,158; 6,268,218; 6,291,191; and 6,303,322; all of which are incorporated herein by reference in their entireties. Briefly, ThermoFluor® uses a fluorescent probe 1-anilinonaphtalene-8-sulfonate (1,8-ANS) to evaluate the binding affinity of ligands to a protein, independent of its biological function and activity. ThermoFluor® has not been applied to analyze ligand-albumin binding, and its existing condition may not be optimal for analyzing both specificity and affinity. For example, one condition disclosed in U.S. Pat. No. 6,020,141 utilizes about 100 μM of 1,8-ANS. This concentration may not be desirable for simultaneous analysis of specificity and affinity, as specificity analysis generally uses a fluorescent probe present in a less saturated range for a displacement assay to determine an accurate dissociation constant.
Thus, it is the objective of the present application to provide an efficient method for simultaneous analysis of both affinity and specificity of ligand-albumin binding in a high-throughput format.